Anthrax is caused by infection with Bacillus anthracis, a spore-forming, rod-shaped bacterium. The dormant spore-form is highly resistant to extreme conditions, high temperatures, and a variety of chemical treatments. The spores gain entry either through an open wound, causing cutaneous disease, or by ingestion, causing gastrointestinal disease or are inhaled causing inhalation anthrax. All three forms can progress to a systemic infection leading to shock, respiratory failure, and death. (Mock, M. and Mignot, T. (2003), Cell Microbiol., 5(1):15-23). The stability of the spores and their infectious capacity make them a convenient bioterrorist weapon.
The two known toxins of B. anthracis are binary combinations of protective antigen (PA), named for its ability to induce protective immunity against anthrax, with either edema factor (EF) or lethal factor (LF). PA is the cell binding component of both toxins and is responsible for bringing the catalytic EF or LF into the host cells. EF is an adenylate cyclase which converts ATP to cyclic AMP and causes edema (Brossier, F. and Mock, M. (2001), Toxicon. 39(11):1747-55). The combination of PA-EF forms edema toxin (ETx) which causes edema when injected locally. LF is a zinc-dependent endoprotease known to target the amino-terminus of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators (Id.). The cleavage of these proteins disrupts a signaling pathway and leads to cytokine dysregulation and immune dysfunction. LF combined with PA forms lethal toxin (LTx) which is lethal when injected on its own. It is also known that there are fatal anthrax cases where administration of antibiotics and clearance of bacteria have failed to rescue the patient. This indicates that there may be a “point of no return” level of LTx in the blood that may predict the outcome of infection. Clearly, LTx and its components are important targets for diagnostics and quantification.
The protein targets of LF are highly specific, and peptides similar to those proteins can be used as artificial substrates. The inventive method uses an artificial peptide substrate which is clipped by LF producing two distinct smaller peptide products. The LF specific cleaved peptides are detected by a variety of rapid quantitative methods illustratively including matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) that visualizes mass specific peaks of the cleaved peptides and fluorescence resonance energy transfer (FRET) that allows for simple detection using bench top fluorometers. Thus, the instant invention detects the specific activity of LF by measuring peptide cleavage products produced per unit time to indicate the amount of LF present.
Existing assays for LF activity, such as SDS-PAGE (Vitale, G. et al. (2000), Biochem. J., 352:739-745; Duesbery, N. S. et al. (1998), Science, 280, 734-737) or HPLC (Hammond, S. E., Hanna, P. C. (1998), Infect. Immun., 66:2374-2378), are impractical for high-throughput screening of compound collections and rapid diagnosis of host infection. Methods for rapid screening of patients in a hospital setting or identification of potent and selective LF inhibitors requires an assay that is less labor intensive, has faster turnaround, and is effective at low levels of enzyme. (Cummings, R. T. (2002), PNAS, 99:6603-6606).
Development of a safe and effective vaccine for inhalation and other forms of anthrax infection is vital to the health and safety of the population and an essential component of any bioterrorism defense strategy. Additionally, the identification of targeted therapies following anthrax infection is essential to managing a patient population. As such, there exists a need for methods to rapidly identify possible candidate vaccines and treatments. There also exists a need for rapid diagnosis of anthrax infection that can be distinguished from other infections that initially display similar symptoms.